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FIG. 9. Overexpression of Xath5 and NeuroD promotes ectopic activation of Brn3.0 expression. Embryos were injected at the two-cell stage with RNA for ﰁ-galactosidase either alone (A) or in combination with RNA for neuroD (B) or Xath5 (C) and then cultured until stage 14 (neural plate stage). The embryos were stained with Magenta-gal (magenta color) to detect ﰁ-galactosidase expression, followed by whole-mount in situ hybridization using a digoxigenin-labeled Brn-3.0 probe (dark purple). All embryos are shown in a dorsoanterior view with the injected side on the right. (A) A control embryo injected with RNA for ﰁ-galactosidase, showing Brn-3.0 expression as two discrete patches in the anterolateral neural plate. (B) An embryo injected with RNA for neuroD and ﰁ-galactosidase showing an expanded patch of expression of Brn-3.0 in the anterolateral neural plate. (C) An embryo injected with RNA for Xath5 and ﰁ-galactosidase showing general activation of Brn-3.0 expression on the injected side with the most enhanced expression at the anterior of the embryo |
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FIG. 6. Comparison in stage 40 Xenopus retina of X-Notch-1 or neuroD expressions with that of genes involved in cellular determination of retinal cells. (A) Comparison of Xotx2 and neuroD expression patterns. Double in situ hybridizations were performed as in Fig. 1 with Xotx2 in deep purple and neuroD in red. (A) Xotx2 expression was seen in cells in the CMZ and in the central retina, in the outer part of the inner nuclear layer. (B) neuroD labeling was seen in cells in the CMZ and in the central retina, in the outer part of the inner nuclear layer and the outer nuclear layer. C and D show a higher magnification of the CMZ from the sections depicted in A and B, respectively. (E) Double labeling demonstrates that neuroD and Xotx2 expressions start at the same level in the CMZ and that they overlap in the outer part of the inner nuclear layer. Scale bar in A and B, 100 ﰂm; scale bar in C, D, and E, 30 um. (F) Comparison of X-Notch-1 and Brn-3.0 expression patterns. Double in situ hybridizations were performed as in Fig. 1 with Brn-3.0 in deep purple and X-Notch-1 in red. (F) Brn-3.0 is only expressed in the ganglion cell layer. (G) X-Notch-1 labeling is detected in the CMZ except in cells in the most peripheral region. H and I show a higher magnification of the CMZ from the sections depicted in F and G, respectively. (J) Double labeling of retina demonstrates that Brn-3.0 and X-Notch-1 expressions do not overlap. Scale bar in F and G, 100 um; scale bar in H, I, and J, 30 um |
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FIG. 7. Double staining for BrdU uptake and gene expression in stage 40 Xenopus CMZ. (A) Double staining for BrdU uptake and Xath5 expression. Xath5 staining is shown in A, BrdU immunostaining in B. Double staining in C shows that BrdUﰃ cells in the peripheral CMZ are Xath5ﰄ. In the central CMZ, BrdUﰃ cells are stained for Xath5. A few cells are BrdUﰄ and stained with Xath5. (D) Double staining for BrdU uptake and neuroD expression. neuroD staining is shown in D, BrdU immunostaining in E. Double staining in F shows that BrdUﰃ cells in the peripheral CMZ are neuroDﰄ. In the central CMZ, BrdUﰃ cells are stained for neuroD. neuroDﰃ cells in the central retina are BrdUﰄ. (G) Double staining for BrdU uptake and Brn-3.0 expression. Brn-3.0 staining is shown in G, BrdU immunostaining in H. Double staining in I shows that all BrdUﰃ cells in the CMZ are Brn-3.0ﰄ and that all Brn-3.0ﰃ cells are BrdUﰄ. (J) Double staining for BrdU uptake and Pax6 expression. Pax6 staining is shown in J, BrdU immunostaining in K. Double staining in L shows that BrdUﰃ cells in the CMZ express a low level of Pax6 and that cells in the central retina expressing a strong level of Pax6 are BrdUﰄ. Peripheral CMZ is on the left. Scalebar in A, 30 ﰂm. |
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pou4f1 (POU class 4 homeobox 1) gene expression in Xenopus laevis embryos, NF stage 40, as assayed by in situ hybridization. Lateral view of sectioned eye. |
